Insights into the structure and function of membrane-integrated processive glycosyltransferases

Bi, Yunchen; Hubbard, Caitlin; Purushotham, Pallinti; Zimmer, Jochen

doi:10.1016/j.sbi.2015.07.008

Cited by 51 publications

(49 citation statements)

References 74 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cellulose is synthesized by cellulose synthase, CesA (1), a membrane-integrated processive family-2 glycosyltransferase that shares significant similarity with chitin, hyaluronan, and alginate synthases (2). CesA combines two functions: the enzyme polymerizes UDP-activated glucose (UDP-Glc) in an S N 2-like nucleophilic displacement reaction (3) and translocates the growing cellulose polymer across the plasma membrane through a pore formed by its own transmembrane (TM) region.…”

mentioning

confidence: 99%

A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro

Purushotham

Cho

Díaz-Moreno

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme's N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils.biopolymer | cellulose | glycosyltransferase | plant cell wall | membrane transport

show abstract

mentioning

confidence: 99%

A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro

Purushotham

Cho

Díaz-Moreno

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Organisms across all kingdoms of life including some viruses utilize this class of enzymes to secrete some of the most abundant organic molecules on earth (Bi et al, 2015). Some prominent examples include cellulose, a linear polymer of glucose molecules (Fig.…”

Section: Processive Gtsmentioning

confidence: 99%

“…Sugar identities are color coded 1 (Bi et. al., 2015) which comprises a large volume of the extracellular matrix in vertebrates while also being utilized by a number of bacteria and viruses (Hubbard et al, 2012;Bi et al, 2015).…”

Section: Processive Gtsmentioning

confidence: 99%

“…Additionally, Poly-N-acetylglucosamine (PNAG), a homopolymer of β-1,6-linked Nacetylglucosamine sugars, and alginate, a heteropolymer of mannuronic acid and glucuronic acid sugars connected by β-1,4-linkages, are both components of bacterial biofilms, which will be discussed later (Bi et al, 2015).…”

Section: Processive Gtsmentioning

confidence: 99%

“…Some soluble processive GTs have been described such as GlfT2, an enzyme that synthesizes a galactan polymer composed of Bi et. al., 2015) 13 which comprises a large volume of the extracellular matrix in vertebrates while also being utilized by a number of bacteria and viruses (Hubbard et al, 2012;Bi et al, 2015).Additionally, Poly-N-acetylglucosamine (PNAG), a homopolymer of β-1,6-linked Nacetylglucosamine sugars, and alginate, a heteropolymer of mannuronic acid and glucuronic acid sugars connected by β-1,4-linkages, are both components of bacterial biofilms, which will be discussed later (Bi et al, 2015). …”

mentioning

confidence: 99%

See 2 more Smart Citations

Structural Studies of Biopolymer Membrane Transport

Morgan¹

View full text Add to dashboard Cite

The selective transport of specific substrates across the membrane is an essential part of biology.Organisms have evolved a basic mechanism for the transport of ions and small molecule such as sugars across the membrane. This mechanism is termed 'alternating access' and involves the alternating exposure of a substrate-binding site to either side of the membrane. While the molecular details of 'alternating access' have been revealed for a number of different transporters, this scheme seems infeasible for the transport of long biopolymers such as nucleic acids, proteins, and polysaccharides because 'alternating access' requires that the transporter forms a binding site that is large enough to bind the entire substrate. Here, I present novel data addressing the transport mechanism for two bacterial biopolymer transporter proteins: Bacterial Cellulose Synthase which synthesizes and secretes the polysaccharide, cellulose; and PrtD, an ABC transporter which is involved in the secretion of an extracellular protease.Cellulose is a linear polymer of glucose units that forms a major component of plant cell walls as well as many bacterial biofilms. In bacteria, the components required for cellulose biosynthesis are encoded in a single operon minimally made up of the genes bcsA, bcsB, bcsC, and bcsZ. BcsA is an integral inner-membrane (IM) glycosyltransferase enzyme that is responsible for coupling the synthesis of cellulose with its transport across the IM. BcsB is a periplasmic protein with Cterminal IM anchor, and BcsB forms a complex with BcsA (BcsA-B) that is sufficient for in vitro cellulose synthesis. BcsZ is a periplasmic cellulase enzyme, and BcsC is an outer-membrane β-barrel that presumably forms a pore for the cellulose polymer to cross the outer membrane.iii BcsA-B activity is stimulated by the bacterial signaling molecule, cyclic-di-GMP (c-di-GMP), which is a key regulator of biofilm formation. I present crystal structures of the c-di-GMP-bound BcsA-B complex in the presence and absence of UDP, a competitive inhibitor and substrate mimic. The structures reveal that c-di-GMP releases an auto-inhibited state of the enzyme. A salt bridge stabilizes one of the signature c-di-GMP-binding Arg residues in a position to tether a conserved 'gating loop' in front of the active site. The binding of c-di-GMP releases the tether and allows substrate to access the active site. Additionally, the UDP-bound structure reveals an additional role for the 'gating loop' in coordinating substrate at the active site. Functional experiments confirm the structural interpretation by revealing that disruption of the auto-inhibitory salt bridge by mutagenesis generates a constitutively-active cellulose synthase. The mechanistic insights presented here represent the first examples of how c-di-GMP allosterically modulates enzymatic functions.To address the mechanism by which BcsA-B transports cellulose across the IM, I use in crystallo enzymology with the c-di-GMP-bound BcsA-B crystals. Because crystallized BcsA-B is catalytically a...

show abstract

Water aging and the quality of organic carbon sources drive niche partitioning of the active bathypelagic prokaryotic microbiome

Sebastián,

Sánchez,

Salazar

et al. 2024

Limnology & Oceanography

View full text Add to dashboard Cite

Due to the scarcity of organic matter (OM) sources in the bathypelagic (1000–4000 m depth), prokaryotic metabolism is believed to be concentrated on particles originating from the surface. However, the structure of active bathypelagic prokaryotic communities and how it changes across environmental gradients remains unexplored. Using a combination of 16S rRNA gene and transcripts sequencing, metagenomics, and substrate uptake potential measurements, here we aimed to explore how water masses aging and the quality of OM influence the structure of the active microbiome, and the potential implications for community function. We found that the relative contribution of taxa with a free‐living lifestyle to the active microbiome increased in older water masses that were enriched in recalcitrant OM, suggesting that these prokaryotes may also play a substantial role in the bathypelagic metabolism of vast areas of the ocean. In comparison to particle‐associated prokaryotes, free‐living prokaryotes exhibited lower potential metabolic rates, and harbored a limited number of two‐component sensory systems, suggesting they have less ability to sense and respond to environmental cues. In contrast, particle‐associated prokaryotes carried genes for particle colonization and carbohydrate utilization that were absent in prokaryotes with a free‐living lifestyle. Consistently, we observed that prokaryotic communities inhabiting older waters displayed reduced abilities to colonize particles, and higher capabilities to use complex carbon sources, compared to communities in waters with a higher proportion of labile OM. Our results provide evidence of regionalization of the bathypelagic active prokaryotic microbiome, unveiling a niche partitioning based on the quality of OM.

show abstract

Insights into the structure and function of membrane-integrated processive glycosyltransferases

Cited by 51 publications

References 74 publications

A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro

A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro

Structural Studies of Biopolymer Membrane Transport

Water aging and the quality of organic carbon sources drive niche partitioning of the active bathypelagic prokaryotic microbiome

Contact Info

Product

Resources

About